Class eligibility was defined by exposure to contaminated water, a combination of geographic and concentration criteria, and exposure duration. Key criteria included a) exposure to contaminated water from any of six public water districts [two in West Virginia, four in Ohio; see Supplemental Material, Figure 1 (doi:10.1289/ehp.0800379.S1)] or from private water sources within the geographical boundaries of the public water sources which contained ≥ 0.05 ppb PFOA, and b) the ability to document a minimum 12 months of exposure to contaminated water between 1950 and 3 December 2004, at primary residence, place of employment, or school.
Participants supplied documentation demonstrating both their identity and exposure using a combination of Court-defined acceptable documents. Brookmar, Inc. independently verified the authenticity of documents with the issuing agency, and identity documentation was examined to ensure that participants were enrolled only once. Scanned document copies became part of the participant’s Project electronic data record.
Four types of data were collected: a health survey, self-reported anthropometric measurements, a blood sample, and a medical chart review to validate selected self-reported diagnoses. In identifying clinical laboratory tests and selecting diagnoses for validation, priority was given to those with potential associations with PFC exposure as reported in the scientific literature. Clinical laboratory tests included serum lipid, immune, and inflammatory markers; liver, kidney, and thyroid function; complete blood count; serum electrolytes and protein; and endocrine function, including insulin and glucose [see Supplemental Material, Note 4 (doi:10.1289/ehp.0800379.S1)]. Validated medical diagnoses included heart disease, cancers, thyroid disease, neurologic disorders, inflammatory and autoimmune disorders, and pregnancy complications [see Supplemental Material, Note 5 (doi:10.1289/ehp.0800379.S1)].
The health survey gathered demographic data; current and historic residential and employment information, including water source and use; personal medical diagnoses, treatments including medications, and physical symptoms; family medical history; pregnancy history and pregnancy-related outcomes for women; and information about lifestyle and health behaviors. Participants also self-reported their own height, weight, and blood pressure. Brookmar, Inc. contracted with a separate company to independently pilot test the survey, and revisions were made based on pilot-test findings. The final version of the survey was accepted by the settling parties. The survey, a list of the clinical laboratory tests, and the 18 medical diagnoses verified by medical record review are publically available on The C8 Health Project WVU Data Hosting Website (C8 Health Project 2009
An independent information technology (IT) company was contracted to build and manage informatics solutions that addressed Class security concerns and created a web-based mechanism for Project registration and health survey completion. Participants could alternatively register in person and use paper-based surveys. After registration and completion of the health survey, participants received instructions regarding requirements for demonstrating eligibility and making an appointment at a Project data-collection site. Standard data quality-assurance techniques for survey data, including a quota for data duplicate entry for paper-based surveys and electronic-based logic rules (e.g., limited-answer menus) for Web-based surveys, were in place for the health survey.
Data collection procedures
Temporary modular office units were established in each water district, staffed with nurses, phlebotomists, and intake personnel, and equipped for venipuncture, blood processing, and short-term record and blood sample storage. Participants could schedule appointments between 0730 and 1930 hours at the location of their convenience. Because of both feasibility and participant considerations, fasting was not required for phlebotomy, although self-reported fast duration was collected to facilitate interpretation of laboratory results.
At in-person appointments, participants submitted eligibility documentation and the water district indicated by the exposure documentation provided was recorded; this was usually but not always the source of greatest exposure. Project staff verified demographic data, current residential information, and completion of the health survey and asked participants to report their current height, weight, and blood pressure. Participants voluntarily submitted a blood sample.
Each verified participant received $150 for completing the health survey and an additional $250 for providing a blood sample (regardless of sample quantity or quality). The payment amount reflected the compensation intentions of the settlement and remuneration for Project participation expenses.
Blood sample processing and laboratory methods
Blood samples were obtained and processed at individual data collection sites. Samples were drawn into four tubes per participant, with a maximum 35 mL for adults and 26 mL for children. Tubes were spun, aliquotted, and refrigerated until shipping. For limited-volume samples, serum was aliquotted with priority for PFC analysis. Samples were shipped on dry ice daily from each data collection site to the laboratory retained to measure serum PFCs. The clinical laboratory contracted to perform the clinical chemistry analysis picked up samples daily from each data-collection site. Additionally, an aliquot of serum from each participant was frozen and subsequently stored in a Project tissue bank.
Clinical laboratory tests were performed at a large, independent, accredited clinical diagnostic laboratory (LabCorp, Inc., Burlington, NC, USA). A customized health level 7 interface generated immediate, on-site laboratory-specific identification numbers and tube labels and permitted subsequent electronic transfer of clinical laboratory results directly into the Project data system. Clinical laboratory tests and quality assurance were performed in accordance with the accreditation standards required of this laboratory.
The primary laboratory performing PFC analysis (Exygen Research Inc., State College, PA, USA) was selected based on its ability to meet U.S. Food and Drug Administration guidelines for bioanalytical method validation, a lower limit of quantification of 0.5 ng/mL, and 96-well-plate–based technology allowing for high-throughput capability. This was also the laboratory of record for a previously reported, independently performed study of residents in one water district included in the Project (Emmett et al. 2006
). The PFOA quantification and validation methodology used by this laboratory has been previously detailed (Flaherty et al. 2005
). The analytic protocol used for the Project was a modification of this methodology. Briefly, the technique used a protein precipitation extraction together with reverse-phase high-performance liquid chromatography/tandem mass spectrometry. Spectrometric detection was performed using a triple quadrupole mass spectrometer in selected reaction monitoring mode, monitoring for the individual m
transitions for each of the 10 PFCs and the 13
C-PFOA surrogate. Results for the 10 PFCs measured were incorporated into the Project information system through a Windows-based program [see Supplemental Material, Note 6 (doi:10.1289/ehp.0800379.S1)].
PFC quality assurance
A two-tiered quality assurance program was implemented consisting of a
) evaluation of test reliability in the primary lab (intralab reliability) with the use of blank samples, samples spiked with a known PFC concentration, and participant duplicate samples, and b
) use of a second, external laboratory (AXYS Analytical Services Ltd., Sidney, BC, Canada) to determine PFC concentrations for participant duplicate samples (interlab reliability). This laboratory, with the ability to monitor 10 individual PFCs and a lower limit of quantification of 0.2 ng/mL, employed analytic methods previously described (Kuklenyik et al. 2004
; Taniyasu et al. 2005
). Briefly, the technique used solid-phase extraction on a weak anion-exchange column followed by reverse-phase high-performance liquid chromatography/mass spectrometry. Spectrometric detection was performed using a triple quadrupole mass spectrometer in selected reaction monitoring mode, monitoring individual m
transitions for each of the target PFCs, the 13
C-PFOS, and 13
C-PFDA surrogates and the 13
C-PFOA and FOUEA (13
C-2H-perfluoro-2-decenoic acid) instrument internal standards.
To assess method performance at the primary laboratory, quality control samples in the form of two control serum blanks, two lab control spikes in control serum, and two sample duplicates were included with each batch of 90 samples analyzed. 13C-PFOA (surrogate) was also added to every sample before extraction to assess lab preparation. Bulk control blanks and spikes were prepared at the primary lab and sent to the sampling sites. They were then blindly returned with every shipment of samples for analysis to assess storage, transport, and laboratory preparation effects. For these quality control samples, the Project IT system generated in-line dummy identification numbers and two sets of lab-ready, bar-coded phlebotomy tube labels. Site nurses aliquotted two sets of sample tubes, and both were included as part of the standard shipment to the primary laboratory. Based on a data collection site-specific sampling plan, samples were also automatically identified by the Project IT system for the secondary lab. Labels and tubes were generated similarly, as were sample aliquots. Results from quality assurance samples were segregated from the main, participant database post hoc by the IT company, the only group unblinded to identification numbering.
During analysis of quality assurance results, a consistent difference between the primary and secondary laboratory was detected (~ 30%) for samples obtained during the first 4 months of the Project. Investigation and additional, targeted intra- and interlab retesting confirmed these directional (higher) differences. Per a court filing, Exygen discussed the cause as a problem of initially prepared samples used for internal calibration [see Supplemental Material, Note 7 (doi:10.1289/ehp.0800379.S1)]. Affected samples (~ 25,000) were retested using serum stored in the Project tissue bank; quality assurance testing, including sample duplicates and replaced spiked and calibration samples, was also repeated. Retested results demonstrated a consistent decrease from initial results and increased consistency with the secondary lab. All analytic results presented here include only retested values for those affected serum samples.
For quantitative assessment of quality assurance test results, final test values were matched to participant quality assurance values for the primary or secondary lab. Results reported as less than the limit of detection (LOD) were treated conservatively and excluded from quality assurance analyses. Agreement between two measures was assessed with the absolute difference, percent difference (absolute difference between values divided by value means), and coefficient of variation (SD divided by mean), which were then summarized (mean and median) across the matched-samples results.
Validation of select medical diagnoses
Participants self-reporting one of the 18 targeted diagnoses were asked to provide the time and location of diagnosis. After obtaining appropriate record release consent, Brookmar, Inc., requested a copy of supporting documentation from a medical record or pathology report from health care providers. Cooperating providers were compensated $10 per necessary page. For approximately 36,000 validated diagnoses, the following was recorded: self-reported diagnosis; support (confirmation), nonsupport (negation), missing (records not obtained), or substitution (i.e., documentation supported a different diagnosis); the alternate diagnosis where appropriate; and type of documentation used for verification. Review of medical records and determination of diagnostic verification were performed by nurses employed by Brookmar, Inc.
Brookmar, Inc., required that participants read (and “check” affirmatively) an introductory section of the health survey that explained the purpose and procedures of the Project, and risks and benefits of participation. This language is publically available as part of the survey tool. All participants submitting a voluntary blood sample completed the standard consent and release forms of the clinical laboratory contracted for phlebotomy. Brookmar, Inc. obtained a separate consent form for the release of medical records necessary for diagnosis validation, which was subsequently mailed (along with a cover letter and specific documentation request) to the health care provider identified by the participant.
The Project group at West Virginia University and the C8 Science Panel obtained institutional review board (IRB) approval from their own institutions permitting access to deidentified Project data. With assistance from Brookmar, Inc., the C8 Science Panel obtained additional IRB approval allowing access to identified data, which facilitates contacting participants for enrollment in follow-up studies.
After input by the settling parties, Brookmar, Inc. used multiple avenues to publicize the Project. Communications about Project eligibility requirements, enrollment, data collection procedures, time line, and remuneration included a series of open meetings conducted in five of the six water districts, Project Web site messages, a phone bank, and press conferences with local media, an important source of information throughout the duration of the Project.
Brookmar, Inc. also maintained communication with local health care providers. Meetings were conducted to inform the medical community about Project procedures, including medical record requests, as well as the information that participants would be provided pursuant to their Project participation.
Efforts to ensure full access for interested participants included multiple sites, 12-hr daily appointment availability, and disability accommodation including handicap access and assistance completing the health survey. Brookmar, Inc. also coordinated remote data collection for those otherwise eligible Class members unable to travel to a data collection site (e.g., those no longer living in the vicinity). For these participants, eligibility documentation was submitted via mail, followed by remote completion of the health survey and a personal telephone interview. These remote participants then completed phlebotomy at an identified, local, accredited laboratory. Of the total participants, approximately 600 participated via these procedures (Flensborg P, personal communication).
Brookmar, Inc. was unable to accommodate participants cognitively unable to complete the survey (and without a representative to accurately complete it on their behalf) or those physically unable to travel to a phlebotomy site. Thus, these groups are likely underrepresented among Project participants.
Data analysis and reporting
Participants were mailed individualized results for clinical lab tests, including laboratory normal ranges and flags for abnormal findings. For severely abnormal values, emergency flags triggered a personal communication from Brookmar, Inc. personnel with advice to seek prompt medical attention. Participants also received a report of their PFC values.
Upon completion of the Project, Brookmar, Inc. filed an electronic data set with the Wood County Court in May 2008. The data set included the health survey, clinical laboratory and PFC values, an image of eligibility documents, and record of payment. To protect participant privacy, the presiding judge subsequently sealed the data set. A mechanism is currently being sought wherein an agency, likely of the federal government, would maintain and make accessible a deidentified data set for public research use.
The C8 Science Panel is conducting analyses using the Project cross-sectional data collected and is also conducting its own independent, environmental and population-based studies, also financed by the settlement. For consenting participants, the C8 Science Panel is able to link Project-collected data with follow-up and longitudinal studies. A description of ongoing studies is available on the C8 Science Panel’s web site (C8 Science Panel 2009
A tissue bank of participant frozen serum was established at West Virginia University in which samples are stored, handled, and accessed in a manner consistent with the IRB protocol governing the West Virginia University Tissue Bank. The sera can be linked to deidentified Project data and may be used for further studies related to human PFC physiology.
Brookmar, Inc. contracted with the West Virginia University School of Medicine to report to the general public descriptive summaries of results from the Project. Summary data are reviewable at a web site established for that purpose (C8 Health Project 2009
The C8 Science Panel and the West Virginia University Project group are preparing and submitting analyses of associations between PFCs and health outcomes, intended for peer-reviewed journals. These will form part of the evidence that will assist the C8 Science Panel in meeting their Court-appointed obligation to determine “probable links” [see Supplemental Material, Note 3 (doi:10.1289/ehp.0800379.S1)] between PFOA exposure and health outcomes.
In the first phase of data cleaning, the IT company ensured that question responses were consistent with question “skip patterns” and menu options, as well as ensuring consistent coding and formatting for question responses. For the deidentified data set, text fields were scrubbed to eliminate potentially identifying information. In the second phase of data cleaning, completed collaboratively by the C8 Science Panel and West Virginia University Project team, continuous variables were examined and decision rules were created for outliers and missing values. Lab-generated error messages for samples that could not be analyzed were deleted and results set to “null.” For clinical lab results either lower or higher than the LOD, values were replaced with 50% below or above the lower or upper LOD, respectively.
Though serum samples were analyzed for 10 PFCs, not all PFCs were detectable in all samples tested. Four PFCs (PFHS, PFOA, PFOS, PFNA) were detectable in almost all (> 97%) samples; for these PFCs, test results reported as less than the LOD were substituted with 0.25 ng/mL (50% of the lower LOD of 0.5 ng/mL). Three PFCs (PFHxA, PFHpA, PFDA) were detectable in approximately 50% of the samples; results for these PFCs are reported with and/or without substitution for values reported as less than the LOD. Three PFCs (PFPeA, PFUnA, PFDoA) were detectable in only a negligible portion of the tested samples and are not reported here or included in further analyses. Thus, results reported here include 7 of the 10 tested PFCs (PFHS, PFOA, PFOS, PFNA, PFHxA, PFHpA, PFDA).
Estimation of participation rates
Total Project participation was estimated by water district as the number of participating residents divided by the total contemporaneous population in that water district. Water district population was estimated using 2005–2006 Census population estimates for block groups, the smallest Census geographic unit that could be accurately identified. Block groups intersecting with water districts were determined, and the population of each block group was apportioned to the water district based on the ratio of water district area to block group area within each block group. The number of participants resident in each water district was based on the assigned water district, that for which the participant presented qualifying eligibility documents, and self-reported residence at the time of enrollment. Participation estimates for each water district was estimated for age and gender strata.